Probes labeled with energy transfer coupled dyes

Abstract

Fluorescent labels having at least one donor and at least one acceptor fluorophore bonded to a polymeric backbone in energy transfer relationship, as well as methods for their use, are provided. Of particular interest are the subject labels wherein the polymeric backbone is a nucleic acid and the donor fluorophore is bonded to the 5' terminus of said nucleic acid. Such labels find use as primers in applications involving nucleic acid chain extension, such as sequencing, PCR and the like.

What is claimed is:  
     
       1. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label consists of a first specific chemical structure comprising a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first backbone chain, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label consists of a second specific chemical structure comprising a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second backbone chain, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label consists of a third specific chemical structure comprising a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third backbone chain, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label consists of a fourth specific chemical structure comprising a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth backbone chain, with energy transfer from said fourth donor to said fourth acceptor,  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       2. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first backbone chain, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second backbone chain, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third backbone chain, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth backbone chain,  
       provided further that said first donor and said first acceptor are covalently bonded to said first backbone chain to produce a first fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second backbone chain to produce a second fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third backbone chain to produce a third fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth backbone chain to produce a fourth fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said fourth donor to said fourth acceptor, and  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       3. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label is substantially pure and comprises a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first backbone chain, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label is substantially pure and comprises a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second backbone chain, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label is substantially pure and comprises a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third backbone chain, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label is substantially pure and comprises a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth backbone chain, with energy transfer from said fourth donor to said fourth acceptor,  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template produced in said step (a); and  
       (c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       4. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first backbone chain, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second backbone chain, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third backbone chain, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth backbone chain,  
       provided further that said first donor and said first acceptor are covalently bonded to said first backbone chain to produce a substantially pure first fluorescent energy-transfer label with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second backbone chain to produce a substantially pure second fluorescent energy-transfer label with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third backbone chain to produce a substantially pure third fluorescent energy-transfer label with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth backbone chain to produce a substantially pure fourth fluorescent energy-transfer label with energy transfer from said fourth donor to said fourth acceptor, and  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       5. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label consists of a first specific chemical structure comprising a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first covalently bonded chain of atoms, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label consists of a second specific chemical structure comprising a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second covalently bonded chain of atoms, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label consists of a third specific chemical structure comprising a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third covalently bonded chain of atoms, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label consists of a fourth specific chemical structure comprising a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth covalently bonded chain of atoms, with energy transfer from said fourth donor to said fourth acceptor,  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       6. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first covalently bonded chain of atoms, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second covalently bonded chain of atoms, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third covalently bonded chain of atoms, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth covalently bonded chain of atoms,  
       provided further that said first donor and said first acceptor are covalently bonded to said first covalently bonded chain of atoms to produce a first fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second covalently bonded chain of atoms to produce a second fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third covalently bonded chain of atoms to produce a third fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth covalently bonded chain of atoms to produce a fourth fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said fourth donor to said fourth acceptor, and  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       7. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label is substantially pure and comprises a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first covalently bonded chain of atoms, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label is substantially pure and comprises a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second covalently bonded chain of atoms, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label is substantially pure and comprises a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third covalently bonded chain of atoms, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label is substantially pure and comprises a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth covalently bonded chain of atoms, with energy transfer from said fourth donor to said fourth acceptor,  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template produced in said step (a); and  
       (c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       8. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising: 
       (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein:  
       said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine,  
       said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine,  
       said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and  
       said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine,  
       provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first covalently bonded chain of atoms, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second covalently bonded chain of atoms, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third covalently bonded chain of atoms, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth covalently bonded chain of atoms,  
       provided further that said first donor and said first acceptor are covalently bonded to said first covalently bonded chain of atoms to produce a substantially pure first fluorescent energy-transfer label with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second covalently bonded chain of atoms to produce a substantially pure second fluorescent energy-transfer label with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third covalently bonded chain of atoms to produce a substantially pure third fluorescent energy-transfer label with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth covalently bonded chain of atoms to produce a substantially pure fourth fluorescent energy-transfer label with energy transfer from said fourth donor to said fourth acceptor, and  
       further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;  
       (b) separating said different size deoxyribonucleic acids produced in said step (a); and  
       (c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.  
     
     
       9. The method of  claim 1 ,  2 ,  3 , or  4 , wherein said first donor, said second donor, said third donor and said fourth donor are the same. 
     
     
       10. The method of  claim 1 ,  2 ,  3 , or  4  wherein said first backbone chain is derived from a first bi-functional structure, said second backbone chain is derived from a second bi-functional structure, said third backbone chain is derived from a third bi-functional structure, and said fourth backbone chain is derived from a fourth bi-functional structure. 
     
     
       11. The method of  claim 5 ,  6 ,  7 , or  8  wherein said first covalently bonded chain of atoms is derived from a first bi-functional structure, said second covalently bonded chain of atoms is derived from a second bi-functional structure, said third covalently bonded chain of atoms is derived from a third bi-functional structure, and said fourth covalently bonded chain of atoms is derived from a fourth bi-functional structure. 
     
     
       12. The method of  claim 1 ,  2 ,  3 , or  4  wherein said first backbone chain, said second backbone chain, said third backbone chain, and said fourth backbone chain are the same or different. 
     
     
       13. The method of  claim 5 ,  6 ,  7 , or  8  wherein said first covalently bonded chain of atoms, said second covalently bonded chain of atoms, said third covalently bonded chain of atoms, and said fourth covalently bonded chain of atoms are the same or different. 
     
     
       14. The method of  claim 10 , wherein said first bi-functional structure, said second bi-functional structure, said third bi-functional structure, and said fourth bi-functional structure are the same or different. 
     
     
       15. The method of  claim 11 , wherein said first bi-functional structure, said second bi-functional structure, said third bi-functional structure, and said fourth bi-functional structure are the same or different. 
     
     
       16. The method of  claim 10 , wherein said first bi-functional structure comprises two amine functional groups, said second bi-functional structure comprises two amine functional groups, said third bi-functional structure comprises two amine functional groups, and said fourth bi-functional structure comprises two amine functional groups. 
     
     
       17. The method of  claim 11 , wherein said first bi-functional structure comprises two amine functional groups, said second bi-functional structure comprises two amine functional groups, said third bi-functional structure comprises two amine functional groups, and said fourth bi-functional structure comprises two amine functional groups. 
     
     
       18. The method of  claim 10 , wherein said first bi-functional structure comprises a ring structure, said second bi-functional structure comprises a ring structure, said third bi-functional structure comprises a ring structure, and said fourth bi-functional structure comprises a ring structure. 
     
     
       19. The method of  claim 11 , wherein said first bi-functional structure comprises a ring structure, said second bi-functional structure comprises a ring structure, said third bi-functional structure comprises a ring structure, and said fourth bi-functional structure comprises a ring structure. 
     
     
       20. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first fluorescent energy-transfer label is joined to said chain terminator comprising a nitrogenous base complementary to adenine, said second fluorescent energy-transfer label is joined to said second chain terminator comprising a nitrogenous base complementary to guanine, said third fluorescent energy-transfer label is joined to said third chain terminator comprising a nitrogenous base complementary to thymine, and said fourth fluorescent energy-transfer label is joined to said fourth chain terminator comprising a nitrogenous base complementary to cytosine. 
     
     
       21. The method of  claim 20 , wherein said first fluorescent energy-transfer label is joined by a first linking arm to said chain terminator comprising a nitrogenous base complementary to adenine and said second fluorescent energy-transfer label is joined by a second linking arm to said second chain terminator comprising a nitrogenous base complementary to guanine, said third fluorescent energy-transfer label is joined by a third linking arm to said third chain terminator comprising a nitrogenous base complementary to thymine, and said fourth fluorescent energy-transfer label is joined by a fourth linking arm to said fourth chain terminator comprising a nitrogenous base complementary to cytosine. 
     
     
       22. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7  or  8  wherein a first linking arm, a second linking arm, a third linking arm, and a fourth linking arm are the same or different. 
     
     
       23. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first label, said second label, said third label, and said fourth label each have substantially the same mobility under conditions used in said step (b). 
     
     
       24. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first label, said second label, said third label, and said fourth label have a mobility different by not more than 10% under conditions used in said step (b). 
     
     
       25. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first label, said second label, said third label, and said fourth label each have a mobility different by not more than 5% under conditions used in step (b). 
     
     
       26. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first label, said second label, said third label, and said fourth label have the same mobility under conditions used in step (b). 
     
     
       27. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first, second, third, and fourth set of different size nucleic acids complementary to a deoxyribonucleic acid template are produced as a mixture of sets of different size deoxyribonucleic acids. 
     
     
       28. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first fluorescent energy transfer label is FAM-10-FAM and said second fluorescent energy transfer label is FAM-3-ROX. 
     
     
       29. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first fluorescent energy transfer label is FAM-10-FAM, said second fluorescent energy transfer label is FAM-10-JOE, said third fluorescent energy transfer label is FAM-10-TAMRA, and said fourth fluorescent energy transfer label is FAM-10-ROX. 
     
     
       30. The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 , wherein said first fluorescent energy transfer label is FAM-10-FAM, said second fluorescent energy transfer label is FAM-10-JOE, said third fluorescent energy transfer label is FAM-3-TAMRA, and said fourth fluorescent energy transfer label is selected from the group consisting of FAM-3-ROX and FAM-10-ROX.